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Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

Journal of Integrative Agriculture 2024年第1期23卷 228-238页

作者： Wan Wang Zhenjiang Zhang weldu tesfagaber Jiwen Zhang Fang Li Encheng Sun Lijie Tang Zhigao Bu Yuanmao Zhu Dongming Zhao State Key Laboratory for Animal Disease Control and Prevention National African Swine Fever Para-reference LaboratoryNational High Containment Facilities for Animal Diseases Control and PreventionHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China College of Veterinary Medicine Northeast Agricultural UniversityHarbin 150069China

African swine fever(ASF)continues to cause enormous economic loss to the global pig *** there is no safe and effective vaccine,accurate and timely diagnosis of ASF is essential to implement control *** immunofluorescence assay(IFA)is a gold standard serological method recommended by the World Organization for Animal Health(WOAH).In this study,we used primary fetal kidney cells to establish a wild boar cell line(BK2258)that supported the efficient replication of ASF virus(ASFV)SD/DY-I/21 and showed visible cytopathic effect(CPE).Moreover,using BK2258,we established a sensitive and specific IFA for ASFV antibody *** standardize and evaluate the performance of this assay,we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20,and immunized with the vaccine candidate HLJ/18-7GD,field samples,and negative serum *** IFA reacted with the ASFV-positive sera and displayed bright fluorescence *** was no non-specific green fluorescence due to cellular senescence or other cell damage-causing *** to a commercial indirect enzyme-linked immunosorbent assay(iELISA),ASFV antibodies were detected 1–4 days earlier using our *** detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400,respectively,indicating that the IFA is more sensitive than *** newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens(i.e.,Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcme circovirus type 2(PCV2),Pseudorabies virus(PRV),Foot-and-Mouth disease virus type O(FMDV/O),and Porcine epidemic diarrhea virus(PEDV)).This study thus provides a sensitive,specific,and reliable detection method that is suitable for the serological diagnosis of ASF.

关键词： African swine fever antibody IFA serological method

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Developing a duplex ARMS-qPCR method to differentiate genotype Ⅰ and Ⅱ African swine fever viruses based on their B646L genes

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Journal of Integrative Agriculture 2023年第5期22卷 1603-1607页

作者： DING Lei-lei REN Tao HUANG Lian-yu weldu tesfagaber ZHU Yuan-mao LI Fang SUN En-cheng BU Zhi-gao ZHAO Dong-ming State Key Laboratory for Animal Disease Control and Prevention National High Containment Facilities for Animal Diseases Control and PreventionNational African Swine Fever Para-reference LaboratoryHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China College of Veterinary Medicine Xinjiang Agricultural UniversityUrumqi 830052P.R.China

Africanswinefever(ASF),causedbythe African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild *** disease is notifiable and listed by the World Organization for Animal... 详细信息

关键词： swine fever duplex

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Novel P22-monoclonal antibody based blocking ELISA for the detection of African swine fever virus antibodies in serum

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Biosafety and Health 2022年第4期4卷 234-243页

作者： Ghebremedhin Tsegay weldu tesfagaber Yuanmao Zhu Xijun He Wan Wang Zhenjiang Zhang Encheng Sun Jinya Zhang Yuntao Guan Fang Li Renqiang Liu Zhigao Bu Dongming Zhao State Key Laboratory of Veterinary Biotechnology National High Containment Facilities for Animal Diseases Control and PreventionNational African Swine Fever Para-reference LaboratoryHarbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China Department of Veterinary Science Hamelmalo Agricultural CollegeKerenEritrea

African swine fever(ASF)is a highly infectious,transboundary viral disease of domestic and wild pigs,and is currently the most serious threat to world swine production,resulting in significant economic *** the absence of vaccines and treatments,the control of the disease entirely depends on accurate and early diagnosis accompanied by the culling of infected ***,a highly specific and sensitive diagnostic assay is required during an outbreak and surveillance of the *** this study,a highly sensitive,specific,rapid and repeatable P22-monoclonal antibody-based blocking enzyme-linked immunosorbent assay(bELISA)assay was developed for the detection of antibodies against genotype I and II African swine fever viruses(ASFVs).A total of 806 pig serum samples were tested to evaluate the performance of the diagnostic *** determine the PI(percent Inhibition)cut-off value,receiver-operating characteristic(ROC)analysis was *** to the ROC analysis of the data,98.10%specificity and 100%sensitivity were recorded when the threshold cut-off value of PI was established at 47%.In addition,the assay was able to detect ASFV antibodies as early as 9 days post-infection when serum samples from experimentally infected pigs were *** all together,the results of the present study indicated that the P22-mAb based bELISA assay can be used for rapid and accurate detection of antibodies against ASFV,which could play a valuable role in the containment and prevention of ASFV as an alternative to other serological diagnostic ***,this study will assist researchers to further investigate the immunogenic importance of P22 protein in ASFV infection.

关键词： Monoclonal antibodies African swine fever Blocking ELISA Diagnosis P22

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非洲猪瘟病毒p54/E183L蛋白单克隆抗体的制备、鉴定及应用

非洲猪瘟病毒p54/E183L蛋白单克隆抗体的制备、鉴定及应用

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作者： weldu tesfagaber 中国农业科学院

学位级别：硕士

非洲猪瘟（ASF）是一种由非洲猪瘟病毒（ASFV）引起的家猪高致命性出血性病毒性疾病,目前尚无有效疫苗可用。因此,早期、准确地检测是有效控制该疾病的关键。目前,已有几种基于p72、p30和pp62蛋白的ELISA血清学检测方法。p54是ASFV的结... 详细信息

非洲猪瘟（ASF）是一种由非洲猪瘟病毒（ASFV）引起的家猪高致命性出血性病毒性疾病,目前尚无有效疫苗可用。因此,早期、准确地检测是有效控制该疾病的关键。目前,已有几种基于p72、p30和pp62蛋白的ELISA血清学检测方法。p54是ASFV的结构蛋白和高免疫原性蛋白,也被报道为血清学诊断的良好候选蛋白。改进现有的诊断方法和增加一些验证的技术有助于血清学调查。本研究制备了关于ASFV p54蛋白的单克隆抗体并利用其建立诊断方法,为非洲猪瘟的诊断提供重要的参考工具。本研究旨在制备和鉴定p54单克隆抗体,开发一种基于p54蛋白单克隆抗体的非洲猪瘟病毒抗体检测ELISA方法。本研究成功表达并纯化了ASFV结构蛋白p54/E183L,制备了5株针对p54蛋白的单克隆抗体,并对它们进行了进一步的研究。Western blot和IFA结果表明,所制备的单克隆抗体能识别纯化后的蛋白和ASFV感染后的肺泡巨噬细胞（PAM细胞）中的抗原。将p54基因进行截短表达对其进行表位鉴定,从结果可以看出,所制备的p54单克隆抗体中的2A7,2D9,3F2和3D1株能识别p54/E183L（120-184aa）的C端蛋白序列,而4G5株识别N端（53-82aa）蛋白序列。此外,本研究还评估了这5株p54单克隆抗体用于ASFV抗体检测的潜力,并开发了基于2A7的竞争性酶联免疫吸附试验（c ELISA）。为评价该方法的效果,共检测了365份猪血清样本（178份阴性样本和187份阳性样本）,并使用ROC曲线分析确定临界值。ROC结果分析显示,曲线下面积（AUC）为0.982（95%置信区间:96.9%～99.4%）,以20%的抑制率为阈值,灵敏度为92.5%,特异性为98.9%。与其他市售阻断酶联免疫吸附试验结果（kappa值为0.912）比较,该方法对其他猪病原体均无血清学交叉反应。总的来说,新开发的c ELISA为快速、方便的ASFV血清学诊断提供了一种实用的检测方法,当筛查ASFV可能感染时,该方法可作为其它血清学检测方法的替代品。

关键词：非洲猪瘟病毒单克隆抗体表位鉴定竞争ELISA 诊断

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Host protein ABCE1 interacts with the viral phosphoprotein and promotes rabies virus replication

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Biosafety and Health 2020年第3期2卷 157-163页

作者： Xing Liu Jiwen Zhang Fang Li Yibrah Tekle Hagoss weldu tesfagaber Lulu Wang Zilong Wang Dongming Zhao Zhigao Bu State Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China National High Containment Laboratory for Animal Diseases Control and Prevention Harbin 150069China Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonosis Yangzhou UniversityYangzhou 225009China

Rabies virus(RABV)phosphoprotein(P)plays an important role in disrupting host interferon(IFN)-mediated antiviral immune ***1 is known as an RNase L inhibitor that negatively regulates the 2′,5′-oligoadenylate(2-5A)/RNase L antiviral system related to the IFN signaling *** this study,we screened the host factors associated with RABV P protein,while focusing on ABCE1 because of its role in the life cycle of several *** results showed that knockout of ABCE1 in HEK293T cells inhibited RABV *** contrast,the overexpression of ABCE1 in HEK293T cells enhanced RABV ***,the co-immunoprecipitation assay showed that ABCE1 interacted with RABV P *** ABCE1 and RABV P proteins are related to the IFN signaling pathway,we propose that the interaction of viral P protein with ABCE1 leads to the disruption of host antiviral immune *** summary,our research showed that ABCE1 is an important host protein that interacts with viral P protein and regulates RABV ***,the interaction between ABCE1 and RABV P protein may facilitate the viral P protein-mediated disruption of host antiviral immune response.

关键词： Rabies virus ABCE1 Replication Phosphoprotein Interaction

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非洲猪瘟病毒pA104R蛋白单克隆抗体的制备与应用

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中国预防兽医学报 2021年第11期43卷 1214-1218页

作者：王露露皇甫皓月席飞张振江 weldu tesfagaber 张纪文黄炼榆李芳步志高赵东明中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室黑龙江哈尔滨150069

为了给非洲猪瘟的临床诊断技术和实验室研究提供物质基础,本研究以我国首次分离的非洲猪瘟病毒(ASFV)Pig/HLJ/2018株基因组为模板,扩增pA104R基因,并将其克隆至pET-30a载体,构建重组原核表达质粒pET-30a-A104R,表达并纯化重组pA104R蛋白... 详细信息

为了给非洲猪瘟的临床诊断技术和实验室研究提供物质基础,本研究以我国首次分离的非洲猪瘟病毒(ASFV)Pig/HLJ/2018株基因组为模板,扩增pA104R基因,并将其克隆至pET-30a载体,构建重组原核表达质粒pET-30a-A104R,表达并纯化重组pA104R蛋白(rpA104R),以其为抗原免疫BALB/c小鼠制备单克隆抗体(MAb),并采用ELISA、western blot、间接免疫荧光法(IFA)和免疫组化(IHC)对所获得的MAb进行鉴定。结果显示,正确构建p ET-30a-A104R重组质粒,并获得大小约为12 ku的可溶性蛋白。通过间接ELISA方法筛选到2株阳性杂交瘤细胞株为4G2和4E10,并且2株MAb的重链均为IgG1,轻链为Kappa链。Western blot和IFA结果显示,MAb 4G2株能够特异性地识别ASFV pA104R蛋白和病毒感染的猪肺泡巨噬细胞中表达的pA104R蛋白。IHC结果表明,MAb 4G2可以与病毒感染的阳性猪组织发生特异性反应。本研究也为ASFV基因缺失疫苗的开发提供了技术储备。

关键词：非洲猪瘟病毒 pA104R 蛋白印迹法间接免疫荧光单克隆抗体

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